Intercalating dyes which exhibit enhanced fluorescence upon binding to DNA or RNA are a basic tool in molecular and cell biology. In general, intercalating dyes bind noncovalently to DNA through a combination of hydrophobic interactions with the DNA base-pairs and ionic binding to the negatively charged phosphate backbone. The fluorescence of the dye is ideally increased several-fold upon binding to DNA, thereby enabling the detection of small amounts of nucleic acids. Examples of fluorescent noncovalent DNA binding dyes include ethidium bromide which is commonly used to stain DNA in agarose gels after gel electrophoresis, and propidium iodide and Hoechst 33258 which are used in flow cytometry to determine the DNA ploidy of cells.
Fluorescent nucleic acid labelling dyes preferably absorb light between about 300 and 900 nm and preferably have a Stokes shift of at least about 10 nm. Dyes that absorb light in the 500 to 900 nm range are preferred because they are spectrally removed from other components that may be present in a biological sample and because they may be used with inexpensive light sources. Fluorescent dyes that have a high extinction coefficient, a high quantum yield, and significantly enhanced fluorescence when bound to a nucleic acid are also preferred.
Few new dye chromophores were described until the introduction of Thiazole Orange as a reticulocyte stain in 1986. Lee, et al., "Thiazole Orange: A New Dye for Reticulocyte Analysis", Cytometry 1986 7, 508-517. Thiazole Orange is an asymmetric cyanine dye. Although many asymmetric cyanine dyes have been described in the art (e.g., Lincoln, et al., U.S. Pat. No. 3,282,932), Thiazole Orange's fluorescence properties when bound to DNA and RNA and its utility for labelling nucleic acids had not been previously identified. Lee, et al., U.S. Pat. No. 4,957,870. For example, unlike most asymmetric cyanine dyes, Thiazole Orange exhibits a several thousand-fold enhancement in fluorescence upon binding to DNA.
Since the discovery of Thiazole Orange as a nucleic acid dye, several improvements to Thiazole Orange and its trimethine homologs have been developed to provide dyes with tighter binding to DNA and greater water solubility. Xue, et al. U.S. Pat. No. 5,321,130 and Glazer, et al. U.S. Pat. No. 5,312,921. These dyes generally involve a modification to the quinolinium portion of the dye.
A continuing need exists for new and improved dyes for labelling nucleic acids. In particular, a need exists for dyes which exhibit longer wavelengths and significantly enhanced fluorescence when bound to DNA or RNA.